The long-term objectives of this research proposal are to continue to use a multidisciplinary approach to characterize the transmitters and receptors of the cholinergic pain suppression systems of the pedunculopontine tegmental nucleus-to-rostral ventromedial medulla (PPTg- to-RVM) cholinergic pathway and the lumbar spinal cord of rats. Experiments are designed to test the hypothesis that L-arginine/nitric oxide (NO)/cyclic GMP cascades mediate muscarinic cholinergic antinociception. It is proposed that cholinergic agonists stimulate muscarinic M1 receptors on NO synthesizing (NOergic) cells present in the PPTg, RVM, and lumbar spinal cord resulting in the production of NO which then diffuses by volume transmission to activate its specific receptor, guanylyl cyclase. It is predicted that agents which inhibit NO synthesis will decrease, and agents which enhance NO or cyclic GMP production will increase cholinergic antinociception as measured using the hot-plate and tail-flick tests in rats. It is also predicted that the proposed target cells for cholinergic analgesics have M1 receptors and contain NO synthase and m1 mRNA, and that these cells are In close proximity to putative NO target neurons containing guanylyl cyclase mRNA. These experiments utilize neuropharmacologic, immunocytochemical, autoradiographic and molecular biological approaches. The Specific Aims are (i) To determine whether inhibitors of NO and cyclic GMP synthesis decrease, and agents which enhance NO or cyclic GMP production increase the antinociception produced by cholinergic stimulation of the PPTg, RVM and the lumbar spinal cord; (2) To determine whether the PPTg-to-RVM pathway sends cholinergic axons terminating on NOergic cells in the RVM, and whether these NOergic perikarya are in close proximity to cells containing guanylyl cyclase; (3) To determine whether intrathecally administered (+)-cis-methyldioxolan releases serotonin and norepinephrin in the lumbar spinal cord by a NO- dependent mechanism; (4) To determine the locations of M1 and M2 receptors in relation to the cholinergic, NOergic, and guanylyl cyclase-containing neurons in the superficial dorsal horn of the lumbar-spinal cord. This research will increase knowledge of the brain and spinal cord's cholinergic pain suppression systems, and will provide an avenue for the development of new, nonopioid pain interventions and therapeutic agents.